Abstract 750: Calcium-Dependent Tyrosine Kinase PYK2 Plays Crucial Role in Angiotensin II- But Not Norepinephrine-Mediated Activation of Rac/Superoxide Pathway That Differentially Regulates Blood Pressure and Vasoconstriction Unmasked by Analysis of PYK2-Knock-out Mice
Background: Calcium-dependent tyrosine kinase PYK2 is activated with various angiogenic growth factors, including angiotensin II (AngII). We previously reported that PYK2 is involved in AngII-induced activation of Rac/JNK. We newly generated the PYK2-knock-out mice (KO). PYK2-null cells exhibited the impaired cell migration due to dysfunction of PI3K, small GTPase Rho, and Ca(2+)-mobilization.
Methods and Results: Basal blood pressure (BP) measured by tail-cuff method was higher in KO than the wild-type mice (WT) (mean 80±3 vs. 74±3 mmHg, P<0.05). Subcutaneous pump infusion of AngII (1.1mg/kg/day) increased BP in WT, whereas this increase was abolished in KO (mean BP, Day 7: 115±4 vs. 88±5, p<0.05, Day 14: 128±4 vs. 95±8mmHg, p<0.05, n=10 each). Infusion of norepinephrine (NE, 5.6ng/kg/day) increased BP in KO to the level comparable to WT (126±5 vs. 128±8 mmHg). The level of the superoxide in the descending aorta was measured using L-012 chemiluminescence. Although the basal level was similar between WT and KO, AngII infusion elevated the superoxide 6.9-fold in KO (p<0.005, n=7), whereas the elevation in KO was only 2.1-fold (p<0.01, n=7). NE-mediated elevation of superoxide was much smaller than that in AngII and similar between KO and WT (1.7-fold vs. 1.6-fold elevation from the baseline, p<0.05, n=7). Pretreatment with SOD-mimetic, tempol (3mM in drinking water) abolished AngII-mediated BP increase in WT, whereas it did not affect NE-mediated BP response. Tyrosine phosphorylation of PYK2 in the aorta was increased 7 days after AngII infusion (3.2-fold from the baseline, p<0.005, n=5), whereas its phosphorylation was lower by 72% 7 days after NE infusion than AngII-treated mice (P<0.01, n=4). PYK2 was expressed in smooth muscle and endothelial cells in the aorta. Activities of Rho and Rac in Ang-II-infused aorta were evaluated by ‘pull-down assay’. Rho and Rac activities increased 3.2-fold and 3.4-fold in WT, respectively, whereas the increases in KO were only 1.4-fold and 1.3-fold, respectively.
Conclusion: PYK2 plays critical role in Ang-II-mediated, but not norepineprine-mediated activation of Rac/superoxide pathway that differentially regulates blood pressure and vascular function.